Hassan Ishag

Porcine reproductive and respiratory syndrome virus induces autophagy to promote virus replication.

An increasing number of studies demonstrate that autophagy, an intrinsic mechanism that can degrade cytoplasmic components, is involved in the infection processes of a variety of pathogens. It can be hijacked by various viruses to facilitate their replication. In this study, we found that PRRSV infection significantly increases the number of double- or single-membrane vesicles in the cytoplasm of host cells in ultrastructural analysis. Our results showed the LC3-I was converted into LC3-II after virus infection, suggesting the autophagy machinery was activated. We further used pharmacological agents and shRNAs to confirm that autophagy promoted the replication of PRRSV in host cells. Confocal microscopy analysis showed that PRRSV inhibited the fusion between autophagosomes and lysosomes, suggesting that PRRSV induced incomplete autophagy. This suppression caused the accumulation of autophagosomes which may serve as replication site to enhance PRRSV replication. It has been shown that NSP2 and NSP3 of arterivirus are two components of virus replication complex. We also found in our studies that NSP2 colocalized with LC3 in MARC-145 cells by performing confocal microscopy analysis and continuous density gradient centrifugation. Our studies presented here indicated that autophagy was activated during PRRSV infection and enhanced PRRSV replication in host cells by preventing autophagosome and lysosome fusion.

Cellular DDX3 regulates Japanese encephalitis virus replication by interacting with viral un-translated regions.

Abstract Japanese encephalitis virus is one of the most common causes for epidemic viral encephalitis in humans and animals. Herein we demonstrated that cellular helicase DDX3 is involved in JEV replication. DDX3 knockdown inhibits JEV replication. The helicase activity of DDX3 is crucial for JEV replication. GST-pulldown and co-immunoprecipitation experiments demonstrated that DDX3 could interact with JEV non-structural proteins 3 and 5. Co-immunoprecipitation and confocal microscopy analysis confirmed that DDX3 interacts and colocalizes with these viral proteins and viral RNA during the infection. We determined that DDX3 binds to JEV 5′ and 3′ un-translated regions. We used a JEV-replicon system to demonstrate that DDX3 positively regulates viral RNA translation, which might affect viral RNA replication at the late stage of virus infection. Collectively, we identified that DDX3 is necessary for JEV infection, suggesting that DDX3 might be a novel target to design new antiviral agents against JEV or other flavivirus infections.

The DEAD-box RNA helicase DDX5 acts as a positive regulator of Japanese encephalitis virus replication by binding to viral 3′ UTR

Abstract Japanese encephalitis virus (JEV), one of the causes for epidemic encephalitis, belongs to the family of Flaviviridae. In this study, we demonstrated that cellular DEAD-box RNA helicase DDX5 plays an important role in JEV replication. The knockdown of DDX5 was able to decrease JEV replication, and overexpression of DDX5 mutants lacking the helicase activity also reduced JEV replication, suggesting the helicase activity is essential for JEV replication. DDX5 knockdown did not affect virus assembly and release. GST-pulldown and co-immunoprecipitation experiments demonstrated that DDX5 could interact with JEV core protein, non-structural protein 3 (NS3) and 5 (NS5-MTase and NS5-RdRp domains). Meanwhile, we also confirmed that DDX5 interacts with these viral proteins during JEV infection. Confocal microscopy analysis showed that endogenous DDX5 is recruited to the cytoplasm and colocalizes with these viral proteins and viral RNA. RNA-pulldown experiment showed that DDX5 only binds to the JEV 3′ untranslated region (UTR). Finally, we confirmed the role of DDX5 in JEV RNA replication using JEV-replicon system. In conclusion, we identified DDX5 as a positive regulator for JEV replication.

Inhibition of Japanese encephalitis virus infection in vitro and in vivo by pokeweed antiviral protein.

Pokeweed antiviral protein (PAP) is a plant-derived N-glycosidase ribosomal-inactivating protein isolated from Phytolacca americana. The antiviral activity of PAP has been described in several viruses. This study was to investigate the antiviral activity of PAP against Japanese encephalitis virus (JEV) infection in vitro and in vivo. Antiviral activity of PAP against JEV infection was evaluated in vitro using plaque forming assay, qRT-PCR and Western blot analysis. In vitro results showed that PAP inhibited replication of JEV in a dose-dependent manner with 50% inhibitory concentration (IC(50)) of 300 ng/ml (23.1 nM). Depurination assay suggested that the antiviral activity of PAP against JEV infection might be partially due to depurination of JEV genomic RNA. In vivo studies showed that PAP (1.0mg/kg) administered intraperitoneally decreased infection in mice challenged with a lethal dose of JEV, presenting a survival of 87.5% or 85.7% when administered pre-infection or post-infection. Collectively, our studies demonstrated that PAP possesses antiviral activity against JEV infection in vitro and in vivo, providing evidences for further development of PAP as an antiviral agent against JEV infection.

A bursal pentapeptide (BPP-I), a novel bursal-derived peptide, exhibits antiproliferation of tumor cell and immunomodulator activity

The bursa of Fabricius (BF) is the central humoral immune organ unique to birds. Here, we isolated a novel bursal pentapeptide I (BPP-I), LGPGP, from BF. BPP-I could play inhibition effect on MCF-7 but not on CEF or Vero cell proliferation in vitro, and enhance antitumor factor p53 protein expression. Also, BPP-I stimulated antibody production in a dose-dependent manner in hybridoma cell. Furthermore, BPP-I could induce various immune responses in mice immunization experiments, including increase antibody production and cytokines IL-4 and IFN-γ level, and induce T-cell immunophenotyping. These results suggest that BPP-I is a potential immunomodulator of antitumor and immunity. The study could provide some novel insights on the probable candidate reagent for the antitumor and immune improvement.

Design and evaluation of a multi-epitope peptide against Japanese encephalitis virus infection in BALB/c mice.

Epitope-based vaccination is a promising means to achieve protective immunity and to avoid immunopathology in Japanese encephalitis virus (JEV) infection. Several B-cell and T-cell epitopes have been mapped to the E protein of JEV, and they are responsible for the elicitation of the neutralizing antibodies and CTLs that impart protective immunity to the host. In the present study, we optimized a proposed multi-epitope peptide (MEP) using an epitope-based vaccine strategy, which combined six B-cell epitopes (amino acid residues 75-92, 149-163, 258-285, 356-362, 373-399 and 397-403) and two T-cell epitopes (amino acid residues 60-68 and 436-445) from the E protein of JEV. This recombinant protein was expressed in Escherichia coli, named rMEP, and its protective efficacy against JEV infection was assessed in BALB/c mice. The results showed that rMEP was highly immunogenic and could elicit high titer neutralizing antibodies and cell-mediated immune responses. It provided complete protection against lethal challenge with JEV in mice. Our findings indicate that the multi-epitope vaccine rMEP may be an attractive candidate vaccine for the prevention of JEV infection.

Griffithsin binds to the glycosylated proteins (E and prM) of Japanese encephalitis virus and inhibit its infection

Abstract Griffithsin (GRFT) is a broad-spectrum antiviral protein against several glycosylated viruses. In our previous publication, we have shown that GRFT exerted antiviral activity against Japanese encephalitis virus (JEV) infection. Herein, we further elucidated the mechanism by which GRFT inhibits JEV infection in BHK-21 cells. In vitro experiments using Pull-down assay and Co-immunoprecipitation (CO-IP) assay showed that GRFT binds to the JEV glycosylated viral proteins, specifically the enveloped (E) and premature (prM) glycoproteins. The binding of GRFT to the JEV was competitively inhibited by increasing concentrations of mannose; in turns abolished anti-JEV activity of GRFT. We suggested that, the binding of GRFT to the glycosylated viral proteins may contribute to its anti-JEV activity. Collectively, our data indicated a possible mechanism by which GRFT exerted its anti-JEV activity. This observation suggests GRFTs potentials in the development of therapeutics against JEV or other flavivirus infection.

Identification and characterization of novel immunomodulatory bursal-derived pentapeptide-II (BPP-II).

Background: The bursa of Fabricius is the acknowledged central humoral immune organ. Results: Novel bursal-derived BPP-II regulates expressions of immune-related gene and exerts immunomodulatory functions and activates p53 expression with antiproliferation on tumor cells. Conclusion: BPP-II integrates linking between humoral immunity and cell-mediated immunity, including antitumor. Significance: This might be the first sign of mechanisms involved in humoral immune system. The bursa of Fabricius, the acknowledged central humoral immune organ, plays a vital role in B lymphocyte differentiation. However, there are few reports of the molecular basis of the mechanism on immune induction and potential antitumor activity of bursal-derived peptides. In this paper, a novel bursal-derived pentapeptide-II (BPP-II, MTLTG) was isolated and exerted immunomodulatory functions on antibody responses in vitro. Gene microarray analyses demonstrated that BPP-II regulated expression of 2478 genes in a mouse-derived hybridoma cell line. Immune-related gene ontology functional procedures were employed for further functional analysis. Furthermore, the majority of BPP-II-regulated pathways were associated with immune responses and tumor processes. Moreover, BPP-II exhibited immunomodulatory effects on antigen-specific immune responses in vivo, including enhancement of avian influenza virus (H9N2 subtype)-specific antibody and cytokine production and modification of T cell immunophenotypes and lymphocyte proliferation. Finally, BPP-II triggered p53 expression and stabilization and selectively inhibited tumor cell proliferation. These data identified the multifunctional factor, BPP-II, as a novel biomaterial representing an important linking between the humoral central immune system and immune induction, including antitumor. Information generated in this study elucidates further the mechanisms involved in humoral immune system and represents the potential basis of effective immunotherapeutic strategies for treating human tumors and immune improvement.

Deletion of the single putative N-glycosylation site of the porcine circovirus type 2 Cap protein enhances specific immune responses by DNA immunisation in mice

The immunogenicity of a putative N-linked glycosylation site located at amino acids 143-145 (N143YS) of the porcine circovirus 2 (PCV2) Cap protein was investigated. Eukaryotic vectors expressing wild-type PCV2 Cap (pCap) and N-linked glycosylation site mutant Cap (pCap-m) were constructed and the immunogenicity of these proteins was determined following DNA vaccination in BALB/c mice. pCap-m elicited significantly higher Cap-specific T lymphocyte proliferative activity, percentage of CD8(+) T cells, ratio of immunoglobulin (Ig) G2a:IgG1 and levels of interferon-γ compared to pCap (P<0.05). These results indicate that deletion of the N-glycosylation site in the PCV2 Cap protein enhances specific immune responses and may have a role in Cap-based DNA vaccines with enhanced immunogenicity.